Can flowers affect land surface albedo and soil microclimates?

The phenology of vegetation, namely leaf-out and senescence, can influence the Earth's climate over regional spatial scales and long time periods (e.g., over 30 years or more), in addition to microclimates over local spatial scales and shorter time periods (weeks to months). However, the effects of flowers on climate and microclimate are unknown. We investigate whether flowers can influence light reflected by the land surface and soil microclimate in a subalpine meadow. We conducted a flower removal experiment with a common sunflower species, Helianthella quinquenervis, for 3 years (2015, 2017, and 2019). The flower removal treatment simulates the appearance of the meadow when Helianthella flowers earlier under climate change and loses its flowers to frost (other plant structures are not damaged by frost). We test the hypotheses that a reduction in cover of yellow flowers leads to a greener land surface, lower reflectance, warmer and drier soils, and increased plant water stress. Flower removal plots are greener, reflect less light, exhibit up to 1.2 °C warmer soil temperatures during the warmest daylight hours, and contain ca. 1% less soil moisture compared to controls. However, soils were warmer in only 2 of the 3 years, when flower abundance was high. Helianthella water use efficiency did not differ between removal and control plots. Our study provides evidence for a previously undocumented effect of flowers on soil microclimate, an effect that is likely mediated by climate change and flowering phenology. Many anthropogenic environmental changes alter landscape albedo, all of which could be mediated by flowers: climate change, plant invasions, and agriculture. This study highlights how further consideration of the effects of flowers on land surface albedo could improve our understanding of the effects of vegetation on microclimate.

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective.

Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers, in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presented.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate.

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 µg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.

Optoelectronic generation of bio-aqueous femto-droplets based on the bulk photovoltaic effect.

The generation and manipulation of small aqueous droplets is an important issue for nano- and biotechnology, particularly, when using microfluidic devices. The production of very small droplets has been frequently carried out by applying intense local electric fields to the fluid, which requires power supplies and metallic electrodes. This procedure complicates the device and reduces its versatility. In this work, we present a novel and flexible, to the best of our knowledge, electrodeless optoelectronic method for the production of tiny droplets of biologically friendly aqueous fluids. Our method takes advantage of the photoinduced electric fields generated by the bulk photovoltaic effect in iron-doped lithium niobate crystals. Two substrate configurations, presenting the polar ferroelectric axis either parallel or perpendicular to the active surface, have been successfully tested. In both crystal geometries, small droplets on the femtoliter scale have been obtained, although with a different spatial distributions correlated with the symmetry of the photovoltaic fields. The overall results demonstrate the effectiveness of the optoelectronic method to produce femtoliter droplets, both with pure water and with aqueous solutions containing biological material.

Plasmonic Hot-Electron Reactive Oxygen Species Generation: Fundamentals for Redox Biology.

For decades, the possibility to generate Reactive Oxygen Species (ROS) in biological systems through the use of light was mainly restricted to the photodynamic effect: the photoexcitation of molecules which then engage in charge- or energy-transfer to molecular oxygen (O2) to initiate ROS production. However, the classical photodynamic approach presents drawbacks, like per se chemical reactivity of the photosensitizing agent or fast molecular photobleaching due to in situ ROS generation, to name a few. Recently, a new approach, which promises many advantages, has entered the scene: plasmon-driven hot-electron chemistry. The effect takes advantage of the photoexcitation of plasmonic resonances in metal nanoparticles to induce a new cohort of photochemical and redox reactions. These metal photo-transducers are considered chemically inert and can undergo billions of photoexcitation rounds without bleaching or suffering significant oxidative alterations. Also, their optimal absorption band can be shape- and size-tailored in order to match any of the near infrared (NIR) biological windows, where undesired absorption/scattering are minimal. In this mini review, the basic mechanisms and principal benefits of this light-driven approach to generate ROS will be discussed. Additionally, some significant experiments in vitro and in vivo will be presented, and tentative new avenues for further research will be advanced.

Optical Tweezers: Phototoxicity and Thermal Stress in Cells and Biomolecules.

For several decades optical tweezers have proven to be an invaluable tool in the study and analysis of myriad biological responses and applications. However, as with every tool, they can have undesirable or damaging effects upon the very sample they are helping to study. In this review the main negative effects of optical tweezers upon biostructures and living systems will be presented. There are three main areas on which the review will focus: linear optical excitation within the tweezers, non-linear photonic effects, and thermal load upon the sampled volume. Additional information is provided on negative mechanical effects of optical traps on biological structures. Strategies to avoid or, at least, minimize these negative effects will be introduced. Finally, all these effects, undesirable for the most, can have positive applications under the right conditions. Some hints in this direction will also be discussed.

Photothermal effect by 808-nm laser irradiation of melanin: a proof-of-concept study of photothermal therapy using B16-F10 melanotic melanoma growing in BALB/c mice.

The photothermal effect is undergoing great interest due to advances in new photosensitizing materials and better-suited light sources, but studies are frequently hampered by the need to employ exogenous photothermal agents and expensive irradiation devices. Here we present a simple strategy based on direct NIR irradiation of the melanin pigment with a commercial 808-nm laser pointer. Proof-of-concept studies showed efficient photothermal effects on melanin in vitro and in vivo. After NIR irradiation, BALB/c mice bearing B16-F10 melanotic melanoma tumors revealed severe histopathological damage and massive necrosis in melanin-containing tumor tissue, while surrounding healthy tissues showed no damage. Therefore, the feasibility of this approach may allow implementing direct procedures for photothermal therapy of pigmented tumors.

Cell cycle modulation through subcellular spatially resolved production of singlet oxygen via direct 765 nm irradiation: manipulating the onset of mitosis.

Reactive oxygen species, ROS, are acknowledged signaling molecules in cellular processes. Singlet molecular oxygen, O2(a1Δg), is one ROS that can initiate cell responses that range from death to proliferation. To better understand the mechanisms involved, it is necessary to further investigate cell response to the "dose" of O2(a1Δg) that has been selectively produced at the expense of other ROS. In this context, dose refers not just to the amount of O2(a1Δg) produced, but also to the subcellular spatial domain in which it is produced. In this study, we selectively produced small and non-toxic amounts of O2(a1Δg) in sensitizer-free experiments by irradiating oxygen at 765 nm using a laser focused either into the nucleus or cytoplasm of HeLa cells. We find that O2(a1Δg)-mediated cell proliferation depends appreciably on the site of O2(a1Δg) production. At the same incident laser power, irradiation into the cytoplasm elicits moderate enhancement of proliferation, whereas irradiation into the nucleus leads to an appreciable delay in the onset and completion of mitosis. We discuss these results in light of what is known about the intracellular photophysics of O2(a1Δg) and the redox state of different cell domains.

Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants.

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networks.

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described.

NIR laser pointer for in vivo photothermal therapy of murine LM3 tumor using intratumoral China ink as a photothermal agent.

The photothermal effect is one of the most promising photonic procedures currently under development to successfully treat several clinical disorders, none the least some kinds of cancer. At present, this field is undergoing a renewed interest due to advances in both photothermal materials and better-suited light sources. However, scientific studies in this area are sometimes hampered by the relative unavailability of state-of-art materials or the complexity of setting up a dedicated optical facility. Here, we present a simple and affordable approach to do research in the photothermal field that relies on a commercial NIR laser pointer and a readily available everyday pigment: China ink. A proof-of-concept study is presented in which mice bearing intradermal LM3 mammary adenocarcinoma tumors were successfully treated in vivo employing China ink and the laser pointer. TUNEL and Ki-67 post-treatment tissue assessment clearly indicates the deleterious action of the photothermal treatment on the tumor. Therefore, the feasibility of this simple approach has been demonstrated, which may inspire other groups to implement simple procedures to further explore the photothermal effect.

Direct 1O2 optical excitation: A tool for redox biology.

Molecular oxygen (O2) displays very interesting properties. Its first excited state, commonly known as singlet oxygen (1O2), is one of the so-called Reactive Oxygen Species (ROS). It has been implicated in many redox processes in biological systems. For many decades its role has been that of a deleterious chemical species, although very positive clinical applications in the Photodynamic Therapy of cancer (PDT) have been reported. More recently, many ROS, and also 1O2, are in the spotlight because of their role in physiological signaling, like cell proliferation or tissue regeneration. However, there are methodological shortcomings to properly assess the role of 1O2 in redox biology with classical generation procedures. In this review the direct optical excitation of O2 to produce 1O2 will be introduced, in order to present its main advantages and drawbacks for biological studies. This photonic approach can provide with many interesting possibilities to understand and put to use ROS in redox signaling and in the biomedical field.

Switching on a transient endogenous ROS production in mammalian cells and tissues.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Exerting better control and specificity with singlet oxygen experiments in live mammalian cells.

Singlet molecular oxygen, O2(a1Δg), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O2(a1Δg) in this regard is acknowledged, an understanding of the targets and mechanisms of O2(a1Δg) action is inadequate. Thus, methods that better facilitate studies of O2(a1Δg) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O2(a1Δg) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O2(a1Δg) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O2(a1Δg) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O2(a1Δg) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O2(a1Δg) at the expense of other ROS. We also comment on our recent efforts to monitor O2(a1Δg) in cells and to monitor the cell's response to O2(a1Δg).

Establishing the subcellular localization of photodynamically-induced ROS using 3,3'-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration.

The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Reliable Screening of Dye Phototoxicity by Using a Caenorhabditis elegans Fast Bioassay.

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.